1. Field of the Invention
The present invention relates to a vaccine, as well as isolated antigens and antibodies constituting a composition for inhibiting and preventing induced Staphylococcus infection. More particularly the present invention concerns a vaccine for prevent Staphylococcus infection, its prevention by antibody, therapeutic antibodies and production thereof.
2. Description of Related Art
In the past years, an inactivated vaccine comprising hemolysin and coagulase extracted from a culture medium of Staphylococcus aureus was used for treatment of Staphylococcus infections. However, since newly developed antibiotics have also been used for treating Staphylococcus infections, the need for vaccine treatment of Staphylococcus infection has been reduced, and finally the vaccine was no longer used and consigned to lasting oblivion. No development of or ideas for vaccines in this connection have recently appeared. The vaccine for Staphylococcus in the past was not specified as to the effective component, and thus the incomplete combination of purification and undetermined effective component rendered its preventive effect unclear.
Staphylococcus is a Gram-positive bacterium and exists as a normal inhabitant, i.e. indigenous bacterium, of human skin and mucous membrane. Although most strains produce biologically active toxins and enzymes, little is known yet concerning the pathogenesis of Staphylococcal disease which has been markedly aroused in recent years by an apparently increasing incidence of severe illness, particularly in hospital environments where many strains are insensitive to the antimicrobial agents which have so successfully prevented disease mortality by other pyrogenic cocci (Bacterial and Mycotic Infections of Man, 4th Ed, 1991). On the other hand, conventional Staphylococci vaccines have been tried, namely an autogenous vaccine. Although some doctors have tried this vaccine, its efficacy is yet unclear, because the strain has to be isolated from the patient and inactivated by formalin to prepare the vaccine, and the vaccine thus prepared has to be injected over a long period. This vaccination may inhibit the secondary infection in humans, caused by Staphylococci.
For animals, there is a vaccine in which the whole cells and culture broth are separately inactivated with formalin, and thereafter mixed. This vaccine is to prevent mastitis of animals (Watson D. L., Res. Vet. Sci.; 45, 16-21, 1988). For mastitis, the other vaccines are based on highly purified xcex1 and xcex2 toxin (Infect. Immun., 17, 250-256, 1977) and fibronectin-binding protein (FnBP-A) (EUR, 294-349, 1988, Vaccine, 12, 11, 918-922, 1994). It is also known to use a subunit vaccine having components from surface proteins and polysaccharides (T. J. Foster, Vaccine 9, 4, 221-227, 1991), and synthetic ST enterotoxin comprising 4n to 18n amino acids is said to be useful for inducing in vivo antibodies (U.S. Pat. No. 4,499,080). But these are not vaccines for Staphylococcus infection due to the use of only general vaccine antibody inducing agents.
In the present invention, a strong xcex2 hemolytic strain, which is a methicillin resistant strain (MRSA in particular) isolated from patients, is cultured in anaerocolumbia agar with rabbit blood (BBL Japan) by the BBL anaerobic jar system. This strain produces component TSST-1 and strong xcex2 hemolysin. After centrifugation of the cultured broth, the supernatant is filtered, ammonium sulfate is added to a concentration of about 65-85% and the resulting precipitate is preserved for later use. The whole cells are immediately suspended in hypertonic buffer solution and are stirred at a temperature below the denaturing point of protein. After centrifugation, ammonium sulphate is added to the cell extract and the resulting precipitate is mixed with the supernatant precipitate. The mixed solution is treated by ion exchange column chromatography to eliminate impurities. The fraction having molecular weight of about 10,000-70,000 is obtained by gel filtration. The main constituents are TSST-1, hemolysin, and enterotoxin, however, this fraction contains neither endotoxin nor whole cells.
The inactivated vaccine of the present invention is thus based in part on the discovery that the antigens have been isolated by mixing the cell surface extract with the supernatant extract. In addition, the preferred strain is a methicillin resistant bacterium isolated from patients showing excellent antigenic activity; moreover, the antigen derived is extracted from the cell wall using a hypertonic buffer solution under mild conditions, and further the specific antigen includes super antigen, major enterotoxins, and the like. The same enterotoxins, for example A, B and C, are produced not only by Staphylococcus aureus but also by other species of Staphylococcus. The vaccine derived from the other species contains similar compositions as Staphylococcus aureus and may be cross-reactive, and the antibody possesses anti-superantigen neutralizing antibody.
The present invention is therefore directed to a process for producing an antigen preparation for inhibiting or preventing induced Staphylococcal infections in humans, a vaccine containing such an antigen or antigens, and a process for producing antibodies for inhibiting or preventing human and animal infections induced by Staphylococcal compositions containing such antibodies. According to a feature of the present invention, a process for isolating two antigens for preventing or inhibiting human staphylococcal infection comprises separating the whole cells of at least one strain of Staphylococcus from cultured broth, extracting at least some antigens of the cell surface and at least some exotoxin antigens and fractionating the resultant antigen from the extract thus obtained. This vaccine is characterized by containing superantigen (for example TSST-1) and an antibody possessing superantigen neutralizing activity.